PURPOSE To characterize the light-evoked increase in the volume (or hydration) of the subretinal space (SRS) of chick retina and to investigate its retinal pigment epithelial (RPE) origin. METHODS The authors perfused chick retina-RPE-choroid preparations with the extracellular space marker, tetramethylammonium (TMA+), and estimated its concentration ([TMA+]o) using double-barreled, ion-selective electrodes. They recorded dark-light changes in subretinal [TMA+]o and studied the effects of drugs that suppress RPE ion transport and conductances. RESULTS The authors observed a slow, light-evoked decrease in subretinal [TMA+]o, about 7% in amplitude, that slowly returned to baseline and then increased at light OFF. It was 40% to 65% inhibited by bumetanide and furosemide, known to inhibit RPE apical membrane Na-K-Cl cotransport; 70% to 80% inhibited by DIDS, an inhibitor of RPE basal membrane chloride conductance, and 50% inhibited by 0.5 mM BaCl2, a blocker of the K+ conductance of the RPE apical membrane. Decreases in subretinal [TMA+]o also were produced when the authors lowered [Cl-]o or [K+]o in the SRS by reducing concentrations in the retinal perfusate. The effect on [TMA]o was larger for [Cl-]o than for [K+]o and was inhibited by bumetanide and DIDS. CONCLUSIONS Changes in subretinal ion concentrations in light and darkness, most likely [K+]o, modify SRS hydration by modulating RPE apical membrane Na-K-Cl cotransport, as well as the conductive effluxes of K+ across the apical membrane and Cl- across the basal membrane. These changes depend principally on the movement of water into or out of the RPE. In turn, subretinal hydration modifies local ion concentrations and the composition of the interphotoreceptor matrix.